Summary The continued global prevalence of tuberculosis and the increased emphasis on the development of new anti- tuberculosis drugs has resulted in greater clinical trials activities to assess drug efficacies and treatment strategies. However, the amount of time required to perform clinical trials for new anti-tuberculosis drugs and treatments needs to be shortened to allow for acceleration of the drug development pipeline. Current research efforts must be applied to develop biomarkers or biosignatures that predict treatment outcomes much earlier than the conversion of sputum to culture negative after two-months of treatment. The development of biomarkers is also needed to facilitate clinical trials with patients that do not present with the pulmonary form of tuberculosis. Moreover, the continued emergence of drug resistant (including MDR and XDR) strains of Mycobacterium tuberculosis and associated treatment failures underscores the need for biomarker or biosignatures that serve could serve as a prognostic of treatment response. A comparison by liquid chromatography-mass spectrometry (LC/MS) of urine collected from tuberculosis patients at four time points during drug treatment (day-0, two-weeks, one- or two-months, and six-months) has allowed us to develop a signature of over 50 small molecules present in the urine at the time of TB diagnosis, but that significantly dropped in abundance by two weeks of treatment and remained at low levels or disappeared at one or two months of treatment. Similar metabolomic studies with plasma collected from tuberculosis patients in South Africa at day-0 and one-month also demonstrated differences in the metabolic profiles at these two time points. These studies provide proof-of-principal that metabolic fluxes in urine or plasma/sera during the treatment of tuberculosis produce a biosignature of effective treatment or cure. We now propose to expand these studies to address Objective 5 (Development of Diagnostics) of the FDA Office of Critical Path Programs'RFA SF424 RR. More specifically, develop "diagnostic biomarkers that will be reliable surrogates for determination of relapse free cures and prediction of relapse in TB clinical trials". We will synergize our expertise in analytical mass spectrometry and biomarker discovery with the existing resources of clinical trials that target or involve the treatment of tuberculosis patients. These partnerships will provide large data sets of metabolic signatures for validation of existing biomarkers/biosignatures and development of new biosignatures that are not biased by geographical location or underlying conditions such as HIV infection or treatment.